Granadaene Photobleaching Reduces the Virulence and Increases Antimicrobial Susceptibility of Streptococcus agalactiae

Abstract

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is increasingly recognized as a major cause of soft tissue and invasive diseases in the elderly and diabetic populations. Antibiotics like penicillin are used with great frequency to treat these infections, although antimicrobial resistance is increasing among GBS strains and underlines a need for alternative methods not reliant on traditional antibiotics. GBS granadaene pigment is related to the hemolysin/cytolysin of GBS, which is critical for the pathogenesis of GBS diseases. Here, we show that photobleaching granadaene dampens the hemolytic activity of GBS. Furthermore, photobleaching of this antioxidant was found to increase GBS susceptibility to killing by reactive oxygen species like hydrogen peroxide. Treatment with light was also shown to affect GBS membrane permeability and contribute to increased susceptibility to the cell membrane-targeting antibiotic daptomycin. Overall, our study demonstrates dual effects of photobleaching on the virulence and antimicrobial susceptibility of GBS and suggests a novel approach for the treatment of GBS infection.

Figure 1.

Extraction and photobleaching of granadaene pigment within S. agalactiae. (a) Chemical structure of granadaene. (b) Characteristic orange-red color of granadaene extracted from S. agalactiae (ATCC 12386) resuspended in DMSO with 0.1% Trifluoroacetic acid. Absorption spectrum of extracted granadaene exhibits characteristic peaks at 430 nm, 460 nm, 490 nm, and 520 nm. (c) Bleaching efficiency of different wavelengths of nanosecond pulsed light in the bleaching of granadaene pigment solution. Exposure to 60 J/cm² of 430 nm light resulted in greatest decrease in the 490 nm absorption peak from an untreated control. (d) Absorption spectrum of extracted granadaene following exposure to 0 (black), 30 (blue), 60 (red), and 120 J/cm² (yellow) of nanosecond pulsed 430 nm blue light. Absorption peaks initially present at 490 nm disappears upon light exposure. (e) Granadaene-expressing S. agalactiae (NCTC 10/84) exhibits a strong orange-red color when grown on agar plates. (f) Absorption spectrum of S. agalactiae (NCTC 10/84) colonies following exposure to 0 (black), 30 (blue), 60 (red), and 120 J/cm² (yellow) of 430 nm pulsed blue light. Absorption peak for pigment within bacteria appears to shift to 410 nm, which begins to disappear upon light exposure. (g) Raman spectroscopy of S. agalactiae (NCTC 10/84) following exposure to 120 J/cm² of pulsed blue light. Characteristic peaks associated with carotenoid structures at 1126 cm⁻¹ and 1510 cm⁻¹ disappear following light exposure. (h) Photobleaching of granadaene results in visible change in color from orange to white in GBS colonies. Bleaching increases with increasing light exposure. Black wells present within the image are empty.

Figure 2.

Granadaene photobleaching reduces Hemolytic activity in S. agalactiae. (a) Hemolytic assay of overnight cultured GBS treated with 120 J/cm² of 430 nm light alongside untreated GBS sample, a positive 0.1% Triton X control, and a negative PBS control. (b) Percent hemolytic activity of non-exposed and blue light exposed GBS. ****: p < 0.0001.

Figure 3.

H₂O₂ killing assays of wild type and mutant GBS, exhibited through differences in mean bacterial CFU population and standard deviation following incubation within H₂O₂ environment. (a) Pigment expressing GBS exposed to 60 J/cm² of 430 nm nanosecond pulsed light and incubated with 12 mM of H₂O₂ for 1 hour. Combination of 430 nm light exposure and H₂O₂ resulted in eradication of GBS. (b) Pigment deficient ΔCylE GBS exposed to 60 J/cm² of pulsed light and incubated with 12 mM of H₂O₂ for 1 hour. Combination of 430 nm light exposure and H₂O₂ resulted in no significant improvement in H₂O₂ antimicrobial activity. ****: p < 0.0001.

Figure 4.

Granadaene photobleaching facilitates cellular uptake and antimicrobial activity of daptomycin. (a) Membrane permeability assay of GBS exposed to 430 nm nanosecond pulsed light utilizing SYTOX Green. Instantaneous increase in SYTOX Green fluorescence following light exposure indicates immediate membrane permeabilization. (b) Minimum Inhibitory Concentration (MIC) of daptomycin on GBS photobleached with blue light decreases 2-fold from 2 μg/mL to 1 μg/mL. (c) CFU count of time killing assay of GBS exposed to 90 J/cm² of pulsed blue light. GBS exhibits faster response to daptomycin following initial light treatment. (d) CFU count of time killing assay of pigment deficient ΔCylE GBS exposed to 90 J/cm² of pulsed blue light and treated with 10 μg/mL for 8 hours. Light exposed ΔCylE GBS lacks the same daptomycin enhancement observed in the pigmented strain. (e) Confocal imaging of light exposed GBS treated with 30 μg/mL of daptoymcin-BODIPY for 30 minutes. The light treated GBS exhibits greater BODIPY fluorescence compared to the control, indicating higher daptomycin intake. (f) Histogram of signal intensities of GBS bacteria treated with 30 μg/mL of daptoymcin-BODIPY for 30 minutes. Light treated GBS exhibits a greater number of bacteria with higher signal intensities compared to the control.

Figure 5.

Granadaene photobleaching exerts little impact on beta-lactam antibiotic activity on GBS. (a) CFU Time kill assay of GBS exposed to 430 nm pulsed light and treated with 0.2 μg/mL of ampicillin. No significant changes in ampicillin performance observed. (b) CFU Time kill assay of ΔCylE GBS exposed to 430 nm pulsed light and treated with 0.2 μg/mL of ampicillin. No significant changes in ampicillin performance observed. (c) CFU Time kill assay of GBS exposed to 430 nm pulsed light and treated with 0.2 μg/mL of penicillin V. Only minor enhancement of penicillin V activity observed after 6 hours of incubation. (d) CFU Time kill assay of ΔCylE GBS exposed to 430 nm pulsed light and treated with 0.2 μg/mL of penicillin V. Only minor enhancement of penicillin V activity observed after 6 hours of incubation, although not as large as the enhancement observed in the pigment expressing GBS. ****: p < 0.0001; **: p < 0.01.